Transcript profiling in the chl1-5 mutant of Arabidopsis reveals a role of the nitrate transporter NRT1.1 in the regulation of another nitrate transporter, NRT2.1

Abstract

Arabidopsis thaliana mutants deficient for the NRT1.1 NO(3)(-) transporter display complex phenotypes, including lowered NO(3)(-) uptake, altered development of nascent organs, and reduced stomatal opening. To obtain further insight at the molecular level on the multiple physiological functions of NRT1.1, we performed large-scale transcript profiling by serial analysis of gene expression in the roots of the chl1-5 deletion mutant of NRT1.1 and of the Columbia wild type. Several hundred genes were differentially expressed between the two genotypes, when plants were grown on NH(4)NO(3) as N source. Among these genes, the N satiety-repressed NRT2.1 gene, encoding a major component of the root high-affinity NO(3)(-) transport system (HATS), was found to be strongly derepressed in the chl1-5 mutant (as well as in other NRT1.1 mutants). This was associated with a marked stimulation of the NO(3)(-) HATS activity in the mutant, suggesting adaptive response to a possible N limitation resulting from NRT1.1 mutation. However, derepression of NRT2.1 in NH(4)NO(3)-fed chl1-5 plants could not be attributed to lowered production of N metabolites. Rather, the results show that normal regulation of NRT2.1 expression is strongly altered in the chl1-5 mutant, where this gene is no more repressible by high N provision to the plant. This indicates that NRT1.1 plays an unexpected but important role in the regulation of both NRT2.1 expression and NO(3)(-) HATS activity. Overexpression of NRT2.1 was also found in wild-type plants supplied with 1 mM NH(4)(+) plus 0.1 mM NO(3)(-), a situation where NRT1.1 is likely to mediate very low NO(3)(-) transport. Thus, we suggest that it is the lack of NRT1.1 activity, rather than the absence of this transporter, that derepresses NRT2.1 expression in the presence of NH(4)(+). Two hypotheses are discussed to explain these results: (1) NRT2.1 is upregulated by a NO(3)(-) demand signaling, indirectly triggered by lack of NRT1.1-mediated uptake, which overrides feedback repression by N metabolites, and (2) NRT1.1 plays a more direct signaling role, and its transport activity generates an unknown signal required for NRT2.1 repression by N metabolites. Both mechanisms would warrant that either NRT1.1 or NRT2.1 ensure significant NO(3)(-) uptake in the presence of NH(4)(+) in the external medium, which is crucial to prevent the detrimental effects of pure NH(4)(+) nutrition.

Figure 1.

Scatter Plot of Tag Frequencies in Col and chl1-5 SAGE Libraries. The two libraries were obtained from roots of 6-week-old plants grown hydroponically on complete nutrient solution containing 1 mM NH₄NO₃ as N source. A total of 31,354 and 28,451 tags were sequenced for Col-0 and chl1-5, respectively. The tags with no occurrence in one library were set at a copy number of one in this library to enable their representation on a logarithmic scale. The size of the data points is correlated with the number of different tags with the same coordinates. The closed symbols correspond to tags with frequencies significantly different (P < 0.01) between the two libraries.

Figure 2.

Gel Blot Analysis of Transcript Accumulation of Various N Metabolism or Transporter Genes between Roots of Col or chl1-5 Plants. The tag copy numbers of the respective SAGE tags of these genes are indicated on top of the autoradiograms for comparison. The asterisks indicate difference between tag copy number in Col and chl1-5 statistically significant at P < 0.01. The experimental conditions are those described in Figure 1.

Figure 3.

Kinetics of Root ¹⁵NO₃⁻ Influx in 6-Week-Old Col and chl1-5 Plants Grown on Complete Nutrient Solution Containing 1 mM NH₄NO₃ as N Source. Root ¹⁵NO₃⁻ influx was assayed by 5 min labeling in complete nutrient solutions containing ¹⁵NO₃⁻ (99 atom percentage ¹⁵N) at the concentration indicated. (A) ¹⁵NO₃⁻ influx determined after total ¹⁵N analysis in both roots and shoots. (B) Ratio between ¹⁵NO₃⁻ influx in chl1-5 and Col in the low ¹⁵NO₃⁻ concentration range. Values are the mean of 6 to 12 replicates ± se.

Figure 4.

Mapping of the chl1-5 Deletion on F12F1 BAC (Chromosome 1). The structure of the corresponding chl1-5 genomic region was deduced from PCR experiments and sequencing.

Figure 5.

Gel Blot Analysis of NRT2.1 Transcript Accumulation in the Roots of chl1 Mutants. The plants of the various genotypes were grown hydroponically for 5 weeks on complete nutrient solution containing 1 mM NH₄NO₃ as N source and were either kept on this solution or transferred on another one with 1 mM NO₃⁻ as N source 1 week before the harvest. Ler, Landsberg erecta; WS, Wassilewskija.

Figure 6.

Gel Blot Analysis of NRT2.1 Transcript Accumulation in the Roots of Col and chl1-5 Plants in Response to Various N Treatments. The plants were grown hydroponically for 6 weeks on complete nutrient solution containing 1 mM NO₃⁻ as N source. At the time of the experiments, one batch of plants was left on 1 mM NO₃⁻, and others were transferred for various periods to nutrient solutions with different N sources as indicated in the figure. The relative NRT2.1 mRNA levels are the means of the values obtained in two replicate experiments and were determined using 25S as a control.

Figure 7.

Effect of the Presence of NH₄⁺ in the Nutrient Solution on Root ¹⁵NO₃⁻ Influx in Col and chl1-5 Plants. The plants were grown hydroponically for 5 weeks on complete nutrient solution containing 1 mM NH₄NO₃ as N source and were either kept on this solution or transferred on another one with 1 mM NO₃⁻ as N source 1 week before the harvest. Root ¹⁵NO₃⁻ influx was assayed by 5 min labeling at 0.2 mM external ¹⁵NO₃⁻ concentration. Results are the means of eight replicates ± se.

Figure 8.

Gel Blot Analysis of NRT2.1 and AMT1.1 Transcript Accumulation in the Roots of Col and chl1-5 Plants in Response to N Starvation. The plants were grown hydroponically for 6 weeks on complete nutrient solution containing 1 mM NH₄NO₃ as N source before the transfer to an N-deprived medium.

Figure 9.

Gel Blot Analysis of NRT2.1 Transcript Accumulation in the Roots of Col and chl1-5 Plants in Response to the Induction by NO₃⁻ and Day/Night Cycle. The plants were grown hydroponically for 5 (A) or 6 (B) weeks on complete nutrient solution containing 1 mM NH₄NO₃ as N source. The plants used for investigating NRT2.1 induction by NO₃⁻ (A) were transferred for 1 week on an N-deprived medium before the addition of 1 mM NO₃⁻ in the nutrient solution for 6 h. The plants used for investigating the diurnal changes in NRT2.1 expression were harvested either at the end of the night (09 h, closed bar) or at the end of the light period (17 h, open bar) (B).

Figure 10.

Root ¹⁵NH₄⁺ Influx in Col and chl1-5 Plants as a Function of the External ¹⁵NH₄⁺ Concentration. The plants were grown hydroponically for 6 weeks on complete nutrient solution containing 1 mM NH₄NO₃ as N source. Root ¹⁵NH₄⁺ influx was assayed by 5 min labeling at the external ¹⁵NH₄⁺ concentrations indicated. Results are the mean of 8 to 12 replicates ± se.

Figure 11.

Accumulation of Free Amino Acids in the Roots and Shoot of Col and chl1-5 Plants. The plants were grown hydroponically for 6 weeks on complete nutrient solution containing 1 mM NH₄NO₃ as N source. Results are the mean of four replicates ± se.

Figure 12.

Accumulation of NO₃⁻ in the Roots and Shoot of Col and chl1-5 Plants. The plants were grown hydroponically for 5 weeks on complete nutrient solution containing 1 mM NH₄NO₃ as N source and were either kept on this solution or transferred on another one with 1 mM NO₃⁻ as N source 1 week before the harvest. Results are the mean of 12 replicates ± se.

Figure 13.

Accumulation of NO₃⁻ in the Roots and Shoot of Ws and chl1-10 Plants and Gel Blot Analysis of NRT2.1 Transcript Accumulation in the Roots of These Plants as a Function of the External NH₄⁺/NO₃⁻ Ratio. (A) Accumulation of NO₃⁻ in the roots and shoot of Ws and chl1-10 plants. (B) Gel blot analysis of NRT2.1 transcript accumulation in the roots of these plants. The plants were grown hydroponically for 5 weeks on complete nutrient solution containing 1 mM NH₄NO₃ as N source and transferred for 6 d on media containing 1 mM NH₄Cl plus either 0.1, 1, or 10 mM KNO₃. NO₃⁻ accumulation results are the means of 12 replicates ± se.